The proposed research is directed toward understanding how the DNA sequence of a bacterial promoter determines its trancriptional activity. Promoter activities will be measured in vivo using a standardized system in which a DNA restriction fragment containing a promoter is oriented by in vitro recombinant techniques so as to produce expression of an easily assayed gene. The promoters examined will be several well characterized promoters including a number of variations of the trytophan operon promoter. A number of in vitro constructions will be undertaken to provide promoters with specific sequence alterations designed to test models dealing with the relationship of DNA sequence to promoter function. In addition, the response of various promoters to growth conditions and the presence of altered transcriptional proteins will be examined in an effort to establish what aspect of promoter structure is involved in a particular response. STudies of the interaction of RNA polymerase with a number of promoter fragments in vitro are planned to correlate in vivo observations of promoter activity with particular properties (association rate, temperature effect, initiation rate) of the reaction which can be measured in vitro. The structure of the RNA polymerase - promoter DNA complex will be investigated by reacting the DNA in the complex with various reactive agents including antibiotics and determining the segments of promoter DNA which are accessible to these probes. By some knowledge of the role that different parts of the promoter sequence play in specifying the transcription initiation frequence from a given promoter, we will gain some understanding of hw the level of expression of a large number of bacterial operons is determined and coordinated during cell growth.